Creutzfeldt-Jacob Disease (hereinafter abbreviated to “CJD”) is an incurable nervous disease which gives rise to presenile dementia. Once a patient develops the disease, the patient enters a vegetative state, a so-called bedridden state, in about 3 to 5 months, and eventually dies. At present, there is no effective therapy for CJD, and only a symptomatic treatment is provided.
CJD is known not only as a neurodegenerative disease that simply develops mental deterioration but also as a disease that is transmitted from human to human or from animal to animal. Examples of human to human transmission are: the kuru disease, which is considered to be transmitted by eating a human brain; CJD transmitted by a growth hormone preparation; and CJD after dura mater transplantation, which has been recently an issue of concern. New variant CJD (nvCJD), which has been an issue of concern in Britain, is considered to arise from the transmission of bovine spongiform encephalopathy (abbreviated to “BSE” and generally referred to as “mad cow disease”) from bovine to human.
Based on the assumption that the infectious agent of CJD is made of protein, the infectious agent was designated as a “prion” in 1982. In 1985, the prion protein gene that constitutes the infectious agent, i.e., a prion, was cloned. As a result, it was demonstrated that a prion protein was also expressed in a normal animal brain. Also, the deposition of the aberrant prion protein, which was clearly distinguishable from a normal prion protein, was observed in a patient who had developed the disease. At present, in addition to the aforementioned kuru disease and CJD, Gerstmann-Sträussler Syndrome (abbreviated to “GSS”) and fatal familial insomnia (abbreviated to “FFI”) are known as so-called prion diseases that result from prion aberrance in humans.
In the case of humans, a prion protein is composed of 253 amino acids (SEQ ID NO: 2), which are bound to the surface of a cell membrane through glycosyl-phosphatidyl-inositol (GPI) after a signal sequence comprising 22 amino acids at the N terminus and 23 amino acids at the C terminus has been removed (SEQ ID NO: 3).
Meanwhile, variations in the prion protein gene was discovered in 1989, and familial GSS or familial CJD was found to develop with the variation of only one amino acid of the prion protein. The prion protein was found to play an important role in CJD.
The aforementioned nvCJD was reported to develop early in people's lives (in their teens to 30's), and the deposition of the aberrant prion protein is observed not only in the central nervous system, which is known in other forms of CJD, but also in the follicular dendritic cell (FDC) in the lymphoid tissue, which was not observed in other forms of CJD (Hill A. F. et al., Lancet 1997, 349: 99-100). It is not known approximately how many nvCJD carriers exist, and this is a serious issue of concern in Britain to such an extent that it is prohibited to supply blood products prepared from domestic blood.
For one of the reasons, the transmission (infection) of prions within the same species is easy, highly transmissible, and short in the incubation period while the transmission across species requires a long incubation period and is low rate of transmission. Because of this across-species barrier, it was very difficult to detect infectious agents of human prion diseases such as CJD with using an animal model or the like.
For example, an experiment regarding the transmission of the nvCJD to human type transgenic mice has been attempted (Hill, A. F. et al., Nature 1997 , Oct. 2; 389 (6650): 448-50). However, it required a long incubation period (228 days or longer) to develop any symptoms and was not very successful (successful for only 25 out of 56 mice). It has been recently reported that the nvCJD was transmitted in an incubation period of approximately 250 days with the use of bovine-type transgenic mice (Scott M. R. et al., Proc. Natl. Acad. Sci. USA 1999, 96: 15137-42).
A system using transgenic animals has been reported as a method for detecting prions in a sample (JP Patent Publication (Kohyo) No. 11-510496 A (1999)). In an example using mice, the development of scrapie within approximately 50 days has been reported, however, it took approximately 200 days to develop into a human prion disease.
We demonstrated that an aberrant prion protein was deposited in a synapse in the central nervous system of a CJD patient and a CJD-infected mouse using a novel immunostaining procedure that is referred to as the autoclave method (Kitamoto, T. et al., Am. J. Pathol. 140: 1285-1294 (1992); Muramoto, T. et al., Am. J. Pathol. 140: 1411-1420 (1992)). We also found that an aberrant prion protein was deposited in a cell other than one of the central nervous system, i.e., the FDC (Kitamoto, T. et al., J. Virol. 65: 6292-6295 (1991)). The deposition of the aberrant prion protein in the FDC can be detected well before a mouse develops the disease, and thus, a premorbid diagnosis can be performed (Muramoto, T. et al., Am. J. Pathol. 140: 1411-1420 (1992); Muramoto, T. et al., Am. J. Pathol. 143: 1470-1479 (1993)).
Upon the intracranial administration of an infectious agent (mouse prion) to mice, they generally develop the disease in 120 to 140 days. When the deposition of the aberrant prion protein in the FDC is employed as an indication, detection of the infectious agent can be carried out in all the mice after 30 days post administration.
As described above, rapid determination of the prion infection in pharmaceuticals such as blood products requires early detection of infectious agents of human prion diseases such as CJD with using an animal model, or the like.
The deposition of the aberrant prion protein in the FDC was verified only in mice, and it could not be previously detected in human CJD. Due to a species difference between humans and mice, it was known that the deposition of the aberrant prion protein in the FDC was not observed in wild-type mice in the first inoculation for infection from human CJD (Muramoto, T. et al., J. Virol. 67: 6808-6810 (1993)).
Accordingly, a method for directly detecting the aberrance in a human prion protein has been awaited.